Cell culture processes have been developed for the growth of single cell bacteria, yeast and molds, which are resistant to environmental stresses or are encased within a tough cell wall. Mammalian cell culture, however, is much more complex because the cells are delicate and cannot withstand excessive turbulent action without sustaining damage. Moreover, mammalian cell cultures require complex nutrient media and environment to support cell proliferation and growth; and it is frequently required that the cells attach themselves to some substrate surface to remain viable and to duplicate. The particular culture requirements of mammalian cells make successful in vitro culturing of both normal and abnormal (for example, carcinomas) mammalian cells difficult to achieve.
There is a lack of adequate in vitro culture systems which produce mammalian tissue of sufficient size and functionality to allow subsequent study of the tissue or study of the effects of specific compounds or organisms on the tissue. Elaborate culture systems for normal and abnormal mammalian cells have been developed in an attempt to grow tissues, however, most do not mimic in vitro conditions and have many limiting aspects.
Normal mammalian tissue has been grown for limited periods of time as two-dimensional monolayers on gelled substrate or other surface for anchoring the cells. Buset et al. "Defining Conditions to Promote the Attachment of Adult Human Colonic Epithelial Cells", In Vitro Cell. & Dev. Biol., Vol. 23. No. 6 pp. 403-412 (June 1987). Colonic cell cultures surviving longer than 7 weeks have been difficult to achieve since crypt cells are unable to survive standard culture regimens, and two-dimensional organ cultures do not support the de novo assembly of stroma and its contribution to epithelial cell growth. Shamsuddin, "Colon Organ Culture as a Model for Carcinogenesis", Colon Cancer Cells, Moyer and Poste, Eds. Academic Press, Inc. 1990.
To mimic the in situ environment using monolayer culture, cocultures were prepared using two cell types. A "feeder layer" of fibroblasts or other cells supplied the primary cells with nutrients and other factors difficult to incorporate into a substrate and provided the cellular interaction believed to be necessary for the production of differentiated tissue. Reid et al., "Culturing Hepatocytes and Other Differentiated Cells", Hepatology, Vol. 4, No. 3, pp. 548-559 (1984); Haake et al. "Retention of Differentiated Characteristics in Human Fetal Keratinocytes In Vitro", In Vitro Cell. & Dev. Biol., Vol. 25 No. 25 pp. 592-600 (July 1989).
Monolayers "conditioned" with fibroblast cells have been used to impart into the substrate the soluble growth factors for epithelial cells. Kabalin et al. "Clonal Growth of Human Prostatic Epithelial Cells Is Stimulated by Fibroblasts", The Prostate, Vol. 14, pp. 251-263 (1989). Monolayers do not produce a three-dimensional tissue, but rather a two-dimensional spread of cells. Often the cells developed by monolayer culture and coculture become undifferentiated and lack normal function.
Three-dimensional in vitro models of differentiated tissue have been produced, however, the cells often do not demonstrate normal cellular activity. Embryonic avian skeletal muscle cells have been grown in vitro on expandable membranes which are gradually and substantially, continuously stretched to simulate the mechanical stimulation of cells experienced in vivo. U.S. Patent No. 4,940,853, Method for Growing Tissue Specimens in Vitro, Vadenburgh, Jul. 10, 1990. The expandable support membrane supports development of three-dimensional structures more closely resemble tissue grown in vivo, however, normal independent cellular activity has not been identified. Additionally, three-dimensional human mammary epithelial cells have been grown in collagen. U.S. Pat. No. 5,026,637, Soule, et al., Jun. 25, 1991. The cells under the disclosed culture conditions did not undergo terminal differentiation and cell senescence, but rather were "immortal" in that they retained the capacity to divide. Thus, normal cellular activity and naturalization was not observed.
A variety of different cells and tissues, such as bone marrow, skin, liver, pancreas, mucosal epithelium, adenocarcinoma and melanoma, have been grown in culture systems to provide three-dimensional growth in the presence of a pre-established stromal support matrix. U.S. Pat. No. 4,963,489, Three-Dimensional Cell and Tissue Culture System, Naughton, et al., Oct. 16, 1990; U.S. Pat. No. 5,032,508, Three-Dimensional Cell and Tissue Culture System, Naughton, et al., Jul. 16, 1991. A biocompatible, non-living material formed into a three-dimensional structure is inoculated with stromal cells. In some cases, the three-dimensional structure is a mesh pre-coated with collagen. Stromal cells and the associated connective tissue proteins naturally secreted by the stromal cells attach to and envelop the three-dimensional structure. The interstitial spaces of the structure become bridged by the stromal cells, which are grown to at least subconfluence prior to inoculating the three-dimensional stromal matrix with tissue-specific cells.
Similar difficulties experienced with normal cell and tissue cultures have been observed with culture systems for propagating abnormal cells and tissues. Although several human carcinoma cell lines have been propagated in vitro, present in vitro culture systems do not permit reproducible cultures of neoplastic cells in large-scale, three-dimensional configuration. The culture of most neoplastic cells has a low success rate, with low percentages of neoplastic cells being established in vitro. Success in cancer therapy can be greatly enhanced using therapeutic testing in models that closely resemble tumorous tissue in vivo and/or in situ.
High-density, three-dimensional in vitro growth of mammalian tumor cells is problematic due to the effects of shear, turbulence, and inadequate oxygenation in conventional cell culture systems. On a small scale, mammalian tumor cells have been grown in containers with small microwells to provide surface anchors for the cells. However, cell culture processes for mammalian cells in such microwell containers generally do not provide sufficient surface area to grow mammalian cells on a sufficiently large scale basis for many commercial or research applications.
Coculture of tumor and normal cells in solid-state culture has been reported as shown in U.S. Pat. No. 4,352,887, Method and Article for Culturing Differentiated Cells, Reid et al., Oct. 5, 1982. However, the three-dimensional environment and culture did not achieve standard clinical testing protocol, such that the three-dimensional environment is nurtured by a mixed-bed of tumor and normal cells.
It is important that tumor models utilized in vitro mimic in vivo properties of tumor cell lines in order that tumor genesis and tumor cell invasiveness can be observed. Although animal models are useful for studying carcinomas, many biochemical and molecular studies require that cells be grown in vitro. Studies on carcinoma cell lines have centered around the expression of oncogenic and protooncogenic markers and nucleotide sequences in order to elucidate the etiology of malignant transformation. Studies have led to insight and speculation as to the origin of transformation, the genetics of transformation, and the treatment or inhibition of the transformation process. However, the models studied have lacked sufficient fidelity for adequate comparison of in vitro culture systems to observations in situ.
Traditional in vitro tumor models have failed to provide intact cell subpopulations, stable isoenzyme patterns, stable ploidy, stable and broad-based growth patterns, and high-fidelity expression of specific cellular proteins. Large scale, high-fidelity three-dimensional in vitro culture carcinoma models are necessary to studying developmental, mutagenic, metastagenic and transformation properties of carcinomas.
The ability to prepare adequate tissue models will provide an in vivo-like environment for propagating pathogens, which frequently cannot be propagated otherwise without great difficulty. For example, viruses are typically intracellular parasites, and cannot be grown in the laboratory unless the growing medium contains living cells.
Generally, little is known about the mechanism by which a viral infection induces certain changes in the activity of the normal functions of the host organism. For example, much of the material collected and known about Norwalk virus has been obtained from studies of infected volunteers because in vitro systems for cultivation of the virus, as with many other viruses, have not yet been devised. Studies with cultured cell explants often result in the Norwalk virus not producing cytopathic changes in the cells.
Norwalk virus plays a significant role in sporadic illness and in outbreaks of acute nonbacterial gastroenteritis. Kaplan, et al., "Epidemiology of Norwalk Gastroenteritis and the Role of Norwalk Virus in Outbreaks of Acute Nonbacterial Gastroenteritis", Annals of Internal Medicine, Vol. 96 (Part 1), pp. 756-761 (1982). Norwalk infection produces a brief illness characterized primarily by nausea, vomiting, diarrhea, and abdominal cramps. The acute gastroenteritis produced by Norwalk virus is an extraordinarily common, worldwide disease with a significant public health impact. In the United States, it is second in frequency only to acute viral respiratory disease as a disease occurrence in American families. Dolin, et al., "Novel Agents of Viral Enteritis in Humans", The Journal of Infectious Diseases, Vol. 155, No. 3, pp. 365-376 (March 1987). Worldwide, it is estimated that greater than 700 million cases of acute diarrheal disease occur annually in children less than 5 years of age, and such disease may be associated with as many as a 5 million deaths, primarily in developing countries.
Norwalk is a part of the group known as small round structured gastroenteritis viruses (20-30 nm in diameter). Norwalk has a diameter of approximately 27 nm, and has been difficult to successfully propagate in the laboratory. Immunological measurements of susceptibility to Norwalk infection remain unknown because neither humoral nor secretory antibody appears to correlate immunity to infection.
The supply of infectious Norwalk virus material is produced primarily through the production of the disease in volunteers. The infectious material produced provides material for laboratory study of etiologic agents and an experimental host in which important biophysical and immunological properties of etiologic agents can be defined. Blacklow et al., "Acute Infectious Nonbacterial Gastroenteritis: Etiology and Pathogenesis", Annals of Internal Medicine, 76:993-1008 (1972). Such studies also provide the opportunity to study the clinical course and pathogenesis of the disease. These methods of studying a virus, however, pose some risk to the infected volunteers.
Various tissue culture procedures for propagating viruses have been proposed, however, most are tedious and time consuming acute infection procedures. Such procedures require multiple passages of cell free virus in tissue culture before an acceptable virus titer is obtained. The infected cell culture is grown to confluency and the grown cells allowed to age. The aged cells are lysed and the virus extracted. The extracted virus can be used to infect a new cell culture.
A variety of cell cultures can be maintained in monolayers on the smooth surface of a solid support, e.g., Petri dishes, glass bottles, or tubes. As soon as such cultures become confluent, forming a united layer of uniform thickness, a virus may be introduced into the liquid medium covering the cells.
Many types of cells can also be grown in suspension, being dispersed within nutrient medium. A virus may then be propagated in the suspended cells. In the conventional system, the fluid medium is decanted off the suspended cells when peak cell concentration is reached. The cells are resuspended in fresh medium which also carries a virus seed. After the period of virus growth, determined by observations of the cytopathic effect of the cells, the harvest is usually passed through a filter and then through a bacterial sterilizing membrane to obtain a solution which contains free viruses released by the disrupted cells.
A disadvantage of large scale suspended cultures of mammalian cells is that air must be sparged through the medium at a high rate and may damage the cells. Further, the sedimentation of the cells is a time-consuming process, and the sedimented cells may be subjected to a medium environment unfavorable in terms of pH and nutritional factors.
The inability to propagate viruses in cell culture frequently necessitates the use of susceptible sub-human primates to grow a particular virus so as to obtain antigen for diagnostic and therapeutic purposes. For example, sub-human primates may be infected with the hepatitis A virus, the infected liver removed, and used to inoculate an in vitro cell culture. U.S. Pat. No. 4,164,566, Hepatitis A Virus Cell Culture in Vitro, Provost et al., Aug. 14, 1979. The cell culture is incubated until hepatitis A antigen is detectable in the culture cells or fluid. Two serial in vitro passages in cell culture are carried out. The hepatitis A virus so modified can be used to prepare live, attenuated hepatitis A vaccine or an inactivated hepatitis A vaccine. Such practices, however, are impractical for commercial use.
Eucaryotic cells have been cultured within a solid carrier body or bed, which is either porous or in a particulate state to provide sufficient internal cavities or space for immobilizing and growing cells and to allow liquid media to pass through the solid carrier and interact with the cells. U.S. Pat. No. 4,203,801, Cell and Virus Culture Systems, Telling, et al., May 20, 1980. The carrier may consist of natural or synthetic materials, such as silicates of diatomaceous earth or polymer particles. A selected cell line is initiated in a monolayer or suspension culture and propagated until the maximum concentration in the cell culture is achieved. The cells are then passed through the bed, whereby the cells are immobilized in the bed. Medium is added to the system and a seed virus introduced. After the cells are disrupted by the virus infection, virus is released and carried away by the medium. Viral material can then be separated from the medium and is available for vaccine formulation or other uses.
Hepatitis A virus has been propagated in human liver tumor cells grown in petri dishes containing nutrient media until the cells reached confluency. U.S. Pat. No. 4,721,675, Production of Hepatitis A Virus in vitro Utilizing a Persistently Virus Infected Cell Culture System, Chan et al., Jan. 26, 1988. The cells were then infected with a single treatment of an inoculum obtained from a human clinical specimen containing the hepatitis A virus. The cell culture system enabled propagating a persistently infected virus producing cell line for passage to other cultures. The system, however, did not mimic in vivo conditions.
U.S. Pat. No. 5,032,508, Three-Dimensional Cell and Tissue Culture System, Naughton, et al., Jul. 16, 1991, discloses a three-dimensional culture system which can be used to culture a variety of different cells and tissues. The tissue cultures grown were proposed for use as a model system for the study of physiologic or pathologic conditions, such as using mucosal epithelium as a model system to study herpes virus or papillomavirus infection. The tissue cultures disclosed by Naughton are initiated using a composite layer taken from a mesenchymal component. Since single cells are not used, it is difficult to define cellular interstitial matrices. The culture system resembles a two-dimensional culture system and it will be difficult to produce the extra-cellular matrix and interstitial components to allow virus cells to translocate from cell to cell.
Difficulties in propagating the Norwalk virus in vitro has precluded the development of commercially available diagnostic assays. A major obstacle to successfully propagating Norwalk virus in vitro is due to the small amount of virus in the stool available for inoculation. Moreover, suitable animal models for the study of these viruses are not available. Relatively little information is available regarding the biophysical and biochemical characteristics of these agents, and for the most part, their significance as etiologic agents of gastroenteritis remains to be established. Understanding of the pathogenesis and immune responses to these agents is at the early stages, and factors responsible for susceptibility of a viral infection remain poorly understood. Since it is difficult to concentrate the virus inoculum, improved cell lines that mimic normal human small intestine tissue would be invaluable for producing larger quantities of virus.
Three-dimensional normal and abnormal cell aggregates and growth achieved in a microgravity vessel provide a unique system in which to simulate the conditions of clinical therapy in vitro. Normal and abnormal mammalian cells cultured in bioreactors, or microgravity vessels, providing low shear and essentially no relative motion of the culture environment with respect to the walls of the culture vessel grow and proliferate to form three-dimensional tissue masses similar to tissues in vitro. Designs for bioreactors which enable such cell and tissue growth are disclosed in U.S. Pat. Nos. 4,988,623, 5,026,650 and 5,153,131.
The three-dimensional tissue masses produced in the bioreactors in vitro permit inoculating the tissue mass with a pathogen to observe the infection produced by the pathogen under conditions closely resembling those in vivo. The culture system provides for proliferation and appropriate cell maturation to form structures analogous to tissue counterparts in vivo. The system enables replicating a pathogen, such as a virus, having biophysical, morphological, immunological, and biological properties characteristic of the pathogen isolated from human sources. The resulting culture may be used to screen cytotoxin compounds and pharmaceutical compounds in vitro, and to produce biologically active molecules in bioreactors. The use of mircrogravity vessels in studying pathogens such as viruses will aid in understanding the pathogenesis by which viral agents cause illness.
The three-dimensional tissues produced from cells taken from different mammalian systems can serve as a model for therapeutic trials directed toward particular pathogens prior to in vivo experimentation. Pathogen therapies, such as radiation, chemotherapy and drugs, can be tested using pathogen infected three-dimensional tissue masses grown under microgravity conditions. For example, the efficacy of anti-viral medications can be tested using virus infected tissue masses grown in microgravity vessels. The development of a means for culturing viruses will enable performing neutralizing antibody studies. Such studies are critical toward the analysis of host short term and long term immune responses to viruses, such as Norwalk. Viral propagation in a three-dimensional tissue mass grown in a microgravity vessel will provide a means by which host immune response to viral infection can be evaluated. Vaccines could then be evaluated for their ability to induce neutralizing responses.